The invention relates to the field of cancer vaccination and immunotherapy.
A current goal of cancer research is the identification of host factors that either predispose to tumor formation or serve to enhance tumor growth.
Genes that confer the ability to convert cells to a tumorigenic state are known as oncogenes. The transforming ability of a number of retroviruses has been localized in individual viral oncogenes (generally v-onc). Cellular oncogenes (generally c-onc) present in many species are related to viral oncogenes. It is generally believed that retroviral oncogenes may represent escaped and/or partially metamorphosed cellular genes that are incorporated into the genomes of transmissible, infectious agents, the retroviruses.
Some c-onc genes intrinsically lack oncogenic properties, but may be converted by mutation into oncogenes whose transforming activity reflects the acquisition of new properties, or loss of old properties. Amino acid substitution can convert a cellular proto-oncogene into an oncogene. For example, each of the members of the c-ras proto-oncogene family (H-ras, N-ras and K-ras) can give rise to a transforming oncogene by a single base mutation.
Other c-onc genes may be functionally indistinguishable from the corresponding v-onc, but are oncogenic because they are expressed in much greater amounts or in inappropriate cell types. These oncogenes are activated by events that change their expression, but which leave their coding sequence unaltered. The best characterized example of this type of proto-oncogene is c-myc. Changes in MYC protein sequence do not appear to be essential for oncogenicity. Overexpression or altered regulation is responsible for the oncogenic phenotype. Activation of c-myc appears to stem from insertion of a retroviral genome within or near the c-myc gene, or translocation to a new environment. A common feature in the translocated loci is an increase in the level of c-myc expression.
Gene amplification provides another mechanism by which oncogene expression may be increased. Many tumor cell lines have visible regions of chromosomal amplification. For example, a 20-fold c-myc amplification has been observed in certain human leukemia and lung carcinoma lines. The related oncogene N-myc is five to one thousand fold amplified in human neuroblastoma and retinoblastoma. In human acute myeloid leukemia and colon carcinoma lines, the proto-oncogene c-myb is amplified five to ten fold. While established cell lines are prone to amplify genes, the presence of known oncogenes in the amplified regions, and the consistent amplification of particular oncogenes in many independent tumors of the same type, strengthens the correlation between increased expression and tumor growth.
Immunity has been successfully induced against tumor formation by inoculation with DNA constructs containing v-onc genes, or by inoculation with v-onc proteins or peptides. A series of reports describe a form of xe2x80x9chomologousxe2x80x9d challenge in which an animal test subject is inoculated with either v-src oncoprotein or DNA constructs containing the v-src gene. Protective immunity was induced against tumor formation by subsequent challenge with v-src DNA or v-src-induced tumor cells. See, Kuzumaki et al., JNCI (1988), 80:959-962; Wisner et al., J. Virol. (1991), 65:7020-7024; Halpern et al., Virology (1993), 197:480-484: Taylor et al., Virology (1994), 205:569-573; Plachy et al., Immunogenetics (1994), 40:257-265. A challenge is said to be xe2x80x9chomologousxe2x80x9d where reactivity to the product of a targeted gene is induced by immunization with the same gene, the corresponding gene product thereof, or fragment of the gene product. A challenge is xe2x80x9cheterologousxe2x80x9d where reactivity to the product of a targeted gene is induced by immunization with a different gene, gene product or fragment thereof.
WO 92/14756 (1992) describes synthetic peptides and oncoprotein fragments which are capable of eliciting T cellular immunity, for use in cancer vaccines. The peptides and fragments have a point mutation or translocation as compared to the corresponding fragment of the proto-oncogene. The aim is to induce immunoreactivity against the mutated proto-oncogene, not the wild-type proto-oncogene. WO 92/14756 thus relates to a form of homologous challenge.
EP 119,702 (1984) describes synthetic peptides having an amino acid sequence corresponding to a determinant of an oncoprotein encoded by an oncogenic virus, which determinant is vicinal to an active site of the oncoprotein. The active site is a region of the oncoprotein required for oncoprotein function, e.g., catalysis of phosphorylation. The peptides may be used to immunize hosts to elicit antibodies to the oncoprotein active site. EP 119,702 is thus directed to a form of homologous challenge.
The protein product encoded by a proto-oncogene constitutes a self antigen and, depending on the pattern of its endogenous expression, would be tolerogenic at the level of T cell recognition of the self peptides of this product. Thus, vaccination against cancers which derive from proto-oncogene overexpression is problematic.
Recent attempts have been made to induce immunity in vitro or in vivo to the product of the HER-2/neu proto-oncogene. The proto-oncogene encodes a 185-kDa transmembrane protein. The HER-2/neu proto-oncogene is overexpressed in certain cancers, most notably breast cancer. In each report discussed below, the immunogen selected to induce immunity comprised a purified peptide of the p185HER-2/neu protein, and not a cellular immunogen.
Disis et al., Cancer Res. (1994) 54:16-20  identified several breast cancer patients with antibody immunity and CD4+helper/inducer T-cell immunity responses to p185HER-2/neu protein. Antibodies to p185HER-2/neu were identified in eleven of twenty premenopausal breast cancer patients. It was assumed prior to this work that patients would be immununologically tolerant to HER-2/neu as a self-protein and that immunity would be difficult to generate.
Disis et al., Cancer Res. (1994) 54:1071-1076 constructed synthetic peptides identical to p185HER-2/neu protein segments with amino acid motifs similar to the published motif for HLA-A2.1-binding peptides. Out of four peptides synthesized, two were shown to elicit peptide-specific cytotoxic T-lymphocytes by primary in vitro immunization in a culture system using peripheral blood lymphocytes from a normal individual homozygous for HLA-A2. Thus, it was concluded that the p185HER-2/neu proto-oncogene protein contains immunogenic epitopes capable of generating human CD8+ cytotoxic T-lymphocytes.
The cytotoxic T cells elicited in the latter report were not, however, shown to recognize tumor cells, but only targets that bound the synthesized peptides. Other work (Dahl et al., J. Immunol. (1996), 157:239-246) has demonstrated that cytotoxic cells may recognize targets that bind peptide but fail to recognize targets that endogenously synthesize peptide. It is thus unclear whether the cytotoxic cells elicited by Disis et al. would be capable of recognizing tumor cells. In any event, no protection against tumor growth was demonstrated by Disis et al.
Peoples et al., Proc. Natl. Acad. Sci. USA (1995), 92:432-436, report the identification of antigenic peptides presented on the surface of ovarian and breast cancer cells by HLA class I molecules and recognized by tumor-specific cytotoxic T lymphocytes. Both HLA-A2-restricted breast and ovarian tumor-specific cytotoxic T lymphocytes recognized shared antigenic peptides. T cells sensitized against a nine-amino acid sequence of one of the peptides demonstrated significant recognition of HLA-A2 HER2/neu tumors.
It remains unclear whether Peoples et al. have successfully attacked proto-oncogene-encoded self, as the immunizing peptide which is expressed in the tumor cells contained an isoleucine at position 2, whereas the peptide expressed in normal tissue contains valine residue at this position. Moreover, although stimulation of T cells occurred in vitro, this stimulation does not represent a true primary immune response insofar as the starting T cell population represented tumor infiltrating lymphocytes.
The research accounts of Disis et al. and Peoples et al. required a form of in vitro stimulation, either priming as described by Disis et al., or restimulation as described by Peoples et al. The in vitro protocols of Disis et al. and Peoples et al. require a mutant cell line to aid in selection of the peptide which will serve to induce reactivity. Non-mutant, peptide antigen-presenting cells have their HLA class I molecules already loaded with endogenous peptides, a phenomenon which precludes exogenous loading from without. The value of the mutant lines is that they lack the TAP genes (encoding the transporters associated with antigen presentation). Class I binding of internally-derived peptides is significantly lowered, and xe2x80x9cemptyxe2x80x9d class I molecules are present on the cell surface and available for binding of exogenously added peptides. This availability of peptide binding sites on membrane-bound class I allows examination of whether a given peptide will (i) even bind to class I, and (ii) function as a target in cytotoxic T cell assays. However, the need for a mutant cell line for deduction of candidate immunizing peptide sequences limits the usefulness of peptide-based immunization schemes.
Fendly et al., J. Biol. Response Modifiers (1990), 9:449-455 present an account of a polypeptide-based immunotherapy. Purified polypeptide corresponding to the extracellular domain of the p185HER-2/neu protein was obtained from a transfected cell line. The purified peptide was employed in the immunization of guinea pigs. The immunized animals developed a cellular immune response, as monitored by delayed-type hypersensitivity. Antisera derived from immunized animals specifically inhibited the in vitro growth of human breast tumor cells overexpressing p185HER-2/neu. There is no indication by Fendly et al. of induction of self versus non-self reactivity. It is likely that the guinea pigs were chiefly responding to non-self determinants (as defined in terms of the guinea pig host) on the human polypeptide immunogen.
The use of peptides for immunization is of necessity limited to immunization with a single haplotype. There are approximately thirty HLA types in man. In each case of peptide immunization, one must be careful to select peptides which match the host HLA type. The selected peptide must be immunogenic in the host and be capable of presentation to host immune system cells.
What is needed is an immunization method for immunizing humans and animals against self-encoded proto-oncogenes which are associated with the development of cancer, which dispenses with the need for isolating immunogenic, HLA host-matched peptides for immunization.
It is an object of the invention to induce reactivity to self-determinants of the product of an overexpressed proto-oncogene.
It is an object of the invention to provide for a form of therapy or prophylaxis based upon the capacity to induce immune reactivity to proto-oncogene-encoded self as overexpressed in tumor cells.
It is an object of the invention to provide a cellular immunogen for use in immunization against self proto-oncogene determinants.
It is an object of the invention to provide for a method for vaccinating a host against disease associated with the overexpression of a proto-oncogene.
These and other objects will be apparent from the following disclosure.
A method of vaccinating a host against disease associated with the overexpression of a target proto-oncogene is provided. The method comprises:
(a) excising cells from the host;
(b) transfecting the excised cells with at least one transgene construct comprising at least one transgene cognate to the target proto-oncogene and a strong promoter to drive the expression of the transgene in the transfected cells, the transgene encoding a gene product which induces host immunoreactivity to host self-determinants of the product of the target proto-oncogene gene;
(c) returning the excised cells transfected with the transgene construct to the body of the host to obtain expression of the transgene in the host.
According to one principal embodiment of the invention, the transgene comprises wild-type or mutant retroviral oncogene DNA. According to another principal embodiment of the invention, the transgene comprises wild-type or mutant proto-oncogene DNA of a species different from the host species. Where the transgene comprises mutant retroviral oncogene DNA or mutant proto-oncogene DNA, the mutant DNA is preferably nontransforming. The mutant DNA preferably comprises a deletion mutation in a region of the DNA which is essential for transformation. Preferably, the host cells are transfected with a plurality, most preferably at least five, different transgene constructs, each construct encoding a different deletion mutation.
In one preferred embodiment of the invention, the mutant DNA has at least about 75% homology, more preferably at least about 80% homology, most preferably at least about 90% homology, with the corresponding wild-type oncogene or proto-oncogene DNA.
The invention is further directed to a cellular immunogen for immunizing a host against the effects of the product of a target proto-oncogene, the overexpression of which is associated with a cancer. The cellular immunogen comprises the host cells which have been transfected with at least one transgene construct, as described above.
The invention is also directed to a method of preparing the cellular immunogen, by (a) excising cells from the host, and (b) transfecting the excised cells with at least one transgene construct, as described above.
The cells transfected with the transgene are preferably rendered non-dividing prior to return to the body of the host.
The term xe2x80x9ccorresponds toxe2x80x9d is used herein to mean that a polynucleotide sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence.
The term xe2x80x9ccognatexe2x80x9d as used herein refers to a gene sequence that is evolutionarily and functionally related between species. For example but not limitation, in the human genome. the human c-myc gene is the cognate gene to the mouse c-myc gene, since the sequences and structures of these two genes indicate that they are highly homologous and both genes encode proteins which are functionally equivalent.
By xe2x80x9chomologyxe2x80x9d is meant the degree of sequence similarity between two different amino acid sequences, as that degree of sequence similarity is derived by the FASTA program of Pearson and Lipman, Proc. Natl. Acad. Sci. USA (1988), 85:2444-2448, the entire disclosure of which is incorporated herein by reference.
As used herein, the term xe2x80x9coperably linkedxe2x80x9d refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is xe2x80x9coperably linkedxe2x80x9d when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
The word xe2x80x9ctransfectionxe2x80x9d is meant to have its ordinary meaning, that is, the introduction of foreign DNA into eukaryotic cells.
By xe2x80x9ctransgenexe2x80x9d is meant a foreign gene that is introduced into one or more host cells.
By xe2x80x9ctransgene constructxe2x80x9d is meant DNA containing a transgene and additional regulatory DNA, such as promoter elements, necessary for the expression of the transgene in the host cells.